Peter Symonds

Low and high molecular weight poly(L-lysine)s/poly(L-lysine)-DNA complexes initiate mitochondrial-mediated apoptosis differently.

Poly(l‐lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9 kDa), L‐PLL, and high (27.4 kDa), H‐PLL, Mw in free form and DNA‐complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine “scrambling” and loss of membrane integrity, but later (24 h) initiate stress‐induced cell death via mitochondrial permeabilization without the involvement of processed caspase‐2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases‐9 and ‐3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H‐PLL. Cytoplasmic level of Cyt c was more elevated following H‐PLL treatment, but unlike L‐PLL case, inhibition of Bax channel‐forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel‐forming activity had no modulatory effect on L‐PLL‐mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H‐PLL, but not L‐PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H‐PLL and L‐PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL‐mediated late‐phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL‐based transfection vectors. The importance of our data in relation to design of novel and safer cationic non‐viral vectors for human gene therapy is discussed.

Endothelial monocyte-activating polypeptide-II (EMAP-II): a novel inducer of lymphocyte apoptosis

The novel, proinflammatory cytokine endothelial monocyte‐activating polypeptide‐II (EMAP‐II) was first found in tumor cell supernatants. EMAP‐II is closely related or identical to the p43 auxiliary protein of the multisynthase complex, which is involved in protein synthesis. In vitro, EMAP‐II induces procoagulant activity, increased expression of E‐ and P‐selectins and tumor necrosis factor receptor‐1, and ultimately, programmed cell death (apoptosis) in cultured endothelial cells. EMAP‐II is also chemotactic for monocytes and neutrophils. However, the role of the p43/EMAP‐II cytokine form in tumors is not understood. We hypothesized an immune‐regulatory role within neoplastic tissues and investigated its effects on lymphocytes. EMAP‐II causes a dose‐dependent inhibition of proliferation and apoptosis in Jurkat T cells and mitogen‐activated peripheral blood mononuclear cells. Coculture with DLD‐1 colorectal cancer cells or media conditioned by these cells induces apoptosis in Jurkat cells, which is partially reversed by antibodies against EMAP‐II. Our data suggest that EMAP‐II constitutes a component of a novel, immunosuppressive pathway in solid tumors, which is not normally expressed outside the cell but in tumors, may be subject to abnormal processing and released from tumor cells.

Colorectal Cancer Cells Induce Lymphocyte Apoptosis by an Endothelial Monocyte-Activating Polypeptide-II-Dependent Mechanism

Endothelial monocyte-activating polypeptide-II (EMAP-II) was first isolated from cell growth medium conditioned by tumor cells, and is closely related or identical with the p43 component of the mammalian multisynthase complex. In its secreted form, EMAP-II has multiple cytokine-like activities in vitro, inducing procoagulant activity on the surface of endothelial cells, increasing expression of E- and P-selectins and TNF-R1, and directing migration of monocytes and neutrophils. EMAP-II has also been shown to induce apoptosis in endothelial cells, leading to the suggestion that it is a proinflammatory polypeptide with antiangiogenic activity. The role of secreted EMAP-II in tumors remains poorly understood, and we hypothesized that EMAP-II may play a role in immune evasion by tumor cells. We investigated its effects on lymphocytes, using recombinant protein, or colorectal cancer cell lines, as a source of native EMAP-II. Recombinant EMAP-II inhibits DNA synthesis and cell division, and induces apoptosis in mitogen-activated lymphocytes in PBMC preparations, and in Jurkat T cells. Native EMAP-II, released by or expressed on the surface of colorectal carcinoma cells, also induces activation of caspase 8 and apoptosis of PBLs and Jurkat cells, which are partially blocked by addition of Abs against EMAP-II. Thus, activated lymphocytes, along with proliferating endothelial cells, are targets for the cytotoxic activity of EMAP-II. Membrane-bound and soluble EMAP-II appear to play multiple roles in the tumor microenvironment, one of which is to assist in immune evasion.

Citrullinated Vimentin Presented on MHC-II in Tumor Cells Is a Target for CD4+ T-Cell–Mediated Antitumor Immunity

Stressful conditions in the harsh tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. However, autophagy also causes post-translational modification of proteins that are recognized by the immune system. In particular, modified self-antigens can trigger CD4(+) T-cell responses that might be exploited to boost antitumor immune defenses. In this study, we investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. Focusing on the intermediate filament protein vimentin, which is frequently citrullinated in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, we generated citrullinated vimentin peptides for immunization experiments in mice. Immunization with these peptides induced IFNγ- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8(+) T cells. These results show how CD4 cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides may offer especially attractive vaccine targets for cancer therapy.

SCIB2, an antibody DNA vaccine encoding NY-ESO-1 epitopes, induces potent antitumor immunity which is further enhanced by checkpoint blockade.

ABSTRACT Checkpoint blockade has demonstrated promising antitumor responses in approximately 10–40% of patients. However, the majority of patients do not make a productive immune response to their tumors and do not respond to checkpoint blockade. These patients may benefit from an effective vaccine that stimulates high-avidity T cell responses in combination with checkpoint blockade. We have previously shown that incorporating TRP-2 and gp100 epitopes into the CDR regions of a human IgG1 DNA (ImmunoBody®: IB) results in significant tumor regression both in animal models and patients. This vaccination strategy is superior to others as it targets antigen to antigen-presenting cells and stimulates high-avidity T cell responses. To broaden the application of this vaccination strategy, 16 NY-ESO-1 epitopes, covering over 80% of HLA phenotypes, were incorporated into the IB (SCIB2). They produced higher frequency and avidity T cell responses than peptide vaccination. These T cells were of sufficient avidity to kill NY-ESO-1-expressing tumor cells, and in vivo controlled the growth of established B16-NY-ESO-1 tumors, resulting in long-term survival (35%). When SCIB2 was given in combination with Treg depletion, CTLA-4 blockade or PD-1 blockade, long-term survival from established tumors was significantly enhanced to 56, 67 and 100%, respectively. Translating these responses into the clinic by using a combination of SCIB2 vaccination and checkpoint blockade can only further improve clinical responses.

SCIB1, a huIgG1 antibody DNA vaccination, combined with PD-1 blockade induced efficient therapy of poorly immunogenic tumors

Purpose We have previously shown that supraoptimal signaling of high avidity T cells leads to high expression of PD-1 and inhibition of proliferation. This study was designed to see if this effect could be mitigated by combining a vaccine that stimulates high avidity T cells with PD-1 blockade. Experimental Design We investigated the anti-tumor effect of a huIgG1 antibody DNA vaccine (SCIB1) and PD-1 blockade. Results Vaccination of HLA-DR4 transgenic mice with SCIB1 induced high frequency and avidity T cell responses that resulted in survival (40%) of mice with established B16F1-DR4 tumors. SCIB1 vaccination was associated with increased infiltration of CD4 and CD8 T cells within the tumor but was also associated with upregulation of PD-L1 within the tumor environment. PD-1 blockade also resulted in increased CD8 T cell infiltration and an anti-tumor response with 50% of mice showing long term survival. In line with our hypothesis that PD-1/PD-L1 signaling results in inhibition of proliferation of high avidity T cells at the tumor site, the combination of PD-1 blockade with vaccination, enhanced the number and proliferation of the CD8 tumor infiltrate. This resulted in a potent anti-tumor response with 80% survival of the mice. Conclusions There is a benefit in combining PD-1 blockade with vaccines that induce high avidity T cell responses and in particular with SCIB1.

Cell-surface associated p43/endothelial-monocyte-activating-polypeptide-II in hepatocellular carcinoma cells induces apoptosis in T-lymphocytes.

OBJECTIVEnThe novel, proinflammatory cytokine endothelial-monocyte-activating-polypeptide-II (EMAP-II) was first found in tumour cell supernatants and is closely related or identical to the p43 component of the mammalian multisynthetase complex. In its secreted form, EMAP-II has multiple cytokine-like activities in vitro, including chemotactic, procoagulant and antiangiogenic properties. We recently showed that neoplastic but not normal hepatocytes expresses the 34-kDa molecule on the cell surface in vitro and the cell-surface expression is upregulated by treatment with tumour necrosis factor (TNF)-alpha/interferon (IFN)-gamma and/or hypoxia. We hypothesized an immune-regulatory role of EMAP-II within neoplastic tissues and investigated its effects on lymphocytes.nnnMETHODSnTo study the role of EMAP-II in tumour cell-induced lymphocyte killing, Jurkat T-cells were co-cultured with a range of hepatocellular carcinoma (HCC) cell monolayers (HuH-7, HepG2 and Alexander cells), which were either untreated or treated with TNF-alpha/IFN-gamma under normoxic and hypoxic conditions over a period of 16-24 hours. Flow cytometric analysis of apoptosis in Jurkat cells was performed using the annexin-V-FITC/propidium iodide technique.nnnRESULTSnrEMAP-II caused a dose-dependent apoptosis in Jurkat T-cells. Co-culture of Jurkat cells with HCC cell monolayers induced significant apoptosis of the Jurkat cells. In general, under normoxic conditions, cytokine-treated HCC cell monolayer caused more apoptosis than untreated cells. This effect was enhanced by hypoxia. Critically, native EMAP-II expressed on the surface of the HCC cells also induced activation of caspase-8 and apoptosis in Jurkat cells, which was partially but significantly blocked by addition of polyclonal antibodies against EMAP-II to the incubation mixture.nnnCONCLUSIONnOur data suggest that membrane-bound EMAP-II is cytotoxic to lymphocytes and, therefore, might constitute a component of a novel, immunosuppressive pathway by which HCC cells may eliminate attacking T-cells and evade the immune system. The mechanism by which it does so is currently under investigation.

Citrullinated α-enolase is an effective target for anti-cancer immunity

ABSTRACT Targeting post-translationally modified epitopes may provide a new strategy for generating tumor specific immune responses. Citrullination is the post-translational modification of arginine to citrulline catalyzed by peptidylarginine deaminase (PAD) enzymes. Presentation of citrullinated peptides on MHC-II has been associated with autophagy. Tumors upregulate autophagy and present citrullinated peptides in response to stresses including nutrient deprivation, oxygen deprivation, redox stress and DNA damage, making them good targets for immune attack. The ubiquitous glycolytic enzyme α-enolase (ENO1) is often citrullinated and degraded during autophagy. Immunization of mice with two citrullinated ENO1 peptides (ENO1 241–260cit253 or 11–25cit15) induced strong Th1 responses that recognize the post-translationally modified, but not the wild type unmodified epitope. ENO1 11–25cit15 induced tumor therapy of melanoma cells in C57Bl/6 (B16F1 50% survival p = 0.0026) and ENO1 241–260cit253 in HLA-DR4 transgenic mice (B16-DR4 50% survival p = 0.0048). In addition, ENO1 241–260cit253 induced therapy of pancreatic (Pan02-DR4 50% survival p = 0.0076) and lung (LLC/2-DR4 40% survival p = 0.0142) tumors in HLA-DR4 transgenic mice. The unmodified epitope induced no anti-tumor response. Minimal regression of class II negative B16 or LLC/2 tumor was seen, confirming direct recognition of MHC-II was required. Most tumors only express MHC-II in the presence of IFNγ; an IFNγ inducible model showed strong responses, with rejection of tumors in up to 90% of animals (p = 0.0001). In humans, a repertoire to ENO1 241–260cit253 was observed in healthy donors. This response was CD4 mediated and seen in people with a variety of HLA types suggesting a broad application for this vaccine in human cancer therapy.

Abstract A035: Citrullinated α-enolase as a novel target for cancer immunotherapy

Stressful conditions in the tumor microenvironment induce autophagy in cancer cells to promote their survival, however, autophagy also causes post-translational modification of proteins which are recognized by the immune system. In particular, modified self-antigens can trigger CD4+ T cell responses that can be exploited to boost antitumor immune defenses. We have previously investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. These studies showed that vimentin, which is frequently expressed in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, is citrullinated and is a good target for anti-tumor immunity (Ref. 1). Immunization with citrullinated vimentin peptides induced IFNγ- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long term survival in 60-90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8+ T cells. Due to its ubiquitous expression and abundance in most cells, the glycolytic enzyme α-enolase is a protein that is often citrullinated and degraded during autophagy and may represent a further novel antitumor target. In this study we demonstrate that immunization of C57Bl, HLA-DR4 and HLA-DP4 transgenic mice with citrullinated enolase peptides induces strong Th1/cytotoxic CD4 responses that efficiently target tumor cells. The Th1 cell repertoire to citrullinated enolase is also detectable in healthy donors and cancer patients. Immunization of mice with citrullinated enolase peptides led to tumor therapy in HLA-DP4 mice with established B16-DP4 tumors (70% survival p = 0.0058) and in HLA-DR4 transgenic mice with established B16F1-DR4 melanoma (50% survival; p = 0.0048) or Pan02-DR4 pancreatic tumors (survival 50%; p = 0.0076). The response was partially mediated by CD4 cytotoxic T cells as tumor therapy was observed against the HLA-DR4-expressing lung tumor LLC2 (40% survival; p = 0.0142) but no survival advantage was witnessed against LLC2 tumors which do not express class II MHC. As MHC-II is not expressed by the majority of tumors unless induced by IFNγ we designed an HLA-DR4 construct under expression of an IFNγ inducible promoter. Immunization of HLA-DR4 mice with citrullinated enolase peptides led to tumor therapy against the established B16F1-IFNγ inducible DR4 melanoma (90% survival p>0.0001). These results suggest that, similar to citrullinated vimentin, citrullinated α-enolase is a promising novel target for human cancer immunotherapy. References1. Brentville VA, Metheringham RL, Gunn B, Symonds P, Daniels I, Gijon M, Cook K, Xue W, Durrant LG (2016). Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T cell-mediated antitumor immunity. Cancer Research 2016 Feb 1;76(3):548-60. Citation Format: Katherine Cook, Ian Daniels, Victoria Brentville, Rachael Metheringham, Wei Xue, Peter Symonds, Tracy Pitt, Mohammed Gijon, Lindy G. Durrant. Citrullinated α-enolase as a novel target for cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A035.

Abstract A015: Protein arginine deiminase enzymes which citrullinate epitopes for MHC II presentation are independent predictors of survival in colorectal cancer

Citrullination of proteins is associated with a number of autoimmune diseases. Protein Arginine Deiminases (PADs) are a family of Ca2+ dependent enzymes that under cellular stress post-translationally convert arginine to citrulline within protein substrates to generate self-modified neo-antigens. It has been shown that presentation of citrullinated peptides on MHC class II stimulates CD4 T cells to mediate potent anti-tumor immunity (1). In this study we focus on the role of the PADI2 and PADI4 family members in colorectal cancer. Using a tissue microarray of colorectal cancers and compiling a comprehensive database of clinicopathological variables, the expression of PADI2 and PADI4 was assessed by immunohistochemistry in a study cohort. This data was used to correlate PADI2 and PADI4 expression with patient survival. In the study cohort 462 colorectal tumors were stained for PADI2 and PADI4. Of these 171 (37%) and 231 (50%) could not be evaluated for PADI2 and PADI4 staining respectively due to the absence of enough tissue core or no evaluable tumor cells (i.e. all stroma) in the core. Of the 291 evaluable colorectal tumors stained with a PADI2 specific antibody, only 18/291 (6.2%) tumors failed to stain. A further 153/291 (52.5%) stained weakly, 102/291 (35.1%) moderate and 18/291 (6.2%) stained strongly. Of the 231 evaluable colorectal tumors stained with a PADI4 specific antibody, no tumors failed to stain. All cases stained strongly for PADI4 expression within the nucleus. In the cytoplasm 63/231 (27.3%) stained weakly, 143/231 (61.9%) moderate and 25/231 (10.8%) stained strongly. PADI2 expression did not correlate with any clinicopathological variables whereas nuclear but not cytoplasmic PADI4 showed a strong association with histological type (p = 0.008). Kaplan-Meier analysis showed there was a correlation of PADI2 and cytoplasmic PADI4 expression with improved survival. Expression of PADI2 gave an increase in survival time from 44.8 months (95% CI 24.3 to 65.4) to 76.2 months (95% CI 69.9 to 82.4, log rank test, p = 0.012). Expression of cytoplasmic PADI4 increased survival time from 57.9 months (95% CI 43.6 to 72.3) to 77.3 months (95% CI 69.6 to 85.1, log rank test, p = 0.012). No significant correlation was observed between PADI2 and the cytoskeletal protein Vimentin or the glycolytic enzyme α-enolase both reported to be citrullinated by PAD enzymes. PADI2 expression was significantly associated with expression of the Nuclear antigen Ki67 (p = 0.046) a cellular marker for proliferation. Nuclear PADI4 significantly correlated with the cytoplasmic glycolytic enzyme α-enolase only (p = 0.001) and cytoplasmic PADI4 was highly significantly associated with α-enolase located in both the cytoplasm (p References 1. Brentville VA, Metheringham RL, Gunn B, Symonds P, Daniels I, Gijon M, Cook K, Xue W, Durrant LG (2016). Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T cell-mediated antitumor immunity. Cancer Research 2016 Feb 1;76(3):548-60 Citation Format: R. Metheringham, M. Gijon, I. Daniels, K. Cook, P. Symonds, T. Pitt, W. Xue, V. Brentville, L. Durrant. Protein arginine deiminase enzymes which citrullinate epitopes for MHC II presentation are independent predictors of survival in colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A015.